The invention relates to novel oligonucleotides, the process for their preparation and their biological use as mediators in the development of the action of interferons, particularly in the development of a part at least of the antiviral action of interferons.
It is known that interferons constitute a family of proteins characterised particularly by their antiviral properties.
It has been observed that the antiviral effect of interferons is mediated by the synthesis of particular proteins. Specific tests have enabled the function of two of them to be identified, which are both enzymes (BAGLIONI. C., 1979, Interferon induced enzymatic activities and their role in the antiviral state. Cell 17, 255-264).
One of them is a polymerase oligonucleotide (2-5A synthetase). This polymerase oligonucleotide catalyses, after activation by bicatenary RNAs and from ATP, the synthesis of a family of oligonucleotides.
These oligonucleotides are short chains of adenosines connected by phosphodiester linkages 2'.fwdarw.5' (KERR I. M. et BROWN R. E., 1978, pppA2'-p5'A2'p5'A: An inhibitor of protein synthesis synthesized with an enzyme fraction from interferon treated cells. PNAS 75, 256-260) of which the general formula may be represented by pppA (2' p5'A).sub.n. These oligonucleotides may be denoted by "oligonucleotides 2'-5'" particularly "oligoadenylates 2'-5'" or by (2'-5') (A).sub.n. One of these oligoadenylates may be represented by the following formula: ##STR2##
It is composed of short chains containing several adenosine groups (adenine+ribose) joined to one another through phosphodiester linkages, as shown, and in which the position at 5' of the adenine nucleus of the terminal adenosine is linked to a variable number of phosphate groups (up to 3 on the 2'.fwdarw.5' oligoadenylate shown).
When the oligoadenylate 2'.fwdarw.5' is totally desphosphorylated, that is to say when the position at 5' of the adenine nucleus of the terminal adenosine is free from the abovesaid variable number of phosphate groups, the resulting compound is denoted by "nucleus(2'.fwdarw.5')A3'," which is an abbreviation for "riboadenylyl (2'.fwdarw.5') riboadenylyl (2'.fwdarw.5') riboadenosine".
The 2'-5' nuclei corresponding to the dephosphorylated (2'-5') oligoadenylates are also called "cores".
In the rest of the description, the 2'-5' oligoadenylates induced in the treated cells by interferon will also be denoted by "unmodified 2'-5' oligoadenylate".
It is accepted that the expression "oligoadenylates 2'.fwdarw.5'" mentioned above and used below will denote also, for convenience of language, the nucleus (2'-5') (A).sub.n partly or entirely dephosphorylated.
The discovery of these 2'.fwdarw.5' oligoadenylates has revealed a novel class of biologically active oligonucleotides, which are assumed to show an important role as mediators of the action of interferon, particularly in the activation of L. endoribionuclease, which is present both in the cells treated by interferon and in those untreated, and in the inhibition of the synthesis of proteins. However the phosphodiester 2'.fwdarw.5' linkages of these adenylates are rapidly cleaved by an enzyme denoted by 2'-phosphodiesterase (cf. the BAGLIONI reference mentioned above).
L endoribonuclease as well as 2-phosphodiesterase are present at levels substantially equal in the treated cells as well as in the cells untreated with interferon.
When the cell is treated with interferon, the concentration of oligonucleotide 2'-5' polymerase increases. Infection by certain viruses of cells so treated results in the production at the viral replication site of NRA bicatenaries activating oligonucleotide 2'-5' polymerase. There results an increase, transitory and possibly localised, at the replication site of the virus, of the concentration of oligoadenylate 2'.fwdarw.5' (Nilsen T. W. et Baglioni C., 1984. Interferon 5, J. Gresser Ed., Academic Press, New York). These oligonucleotides activate themselves by specifically binding therein endoribonuclease L which degrades the viral RNA messengers.
When the interferon is removed from the culture medium, the activity of the oligonucleotide 2'-5' polymerase decreases and the cell loses its antiviral state.
The synthesis of the proteins induced by interferon is transient and, consequently, the cells kept in the tissue cultures do not maintain a high level of these proteins.
In addition, the 2'.fwdarw.5' oligoadenylates induced in the cells treated with interferon exhibit the drawback of having a low metabolic stability. In fact, the unmodified 2'.fwdarw.5' oligoadenylates are, on the one hand, rapidly hydrolysed by a specific phosphodiesterase degrading the molecule progressively from its ribose 2' terminal, on the other hand, are degraded under the action of a phosphatase on the side of the first ribose connected with the variable number of phosphate groups. (Lebleu B. et Content J., 1982, Interferon 3, J. Gresser Ed. Academic Press, New York).
Researches have been undertaken to find similar compounds to the unmodified 2'.fwdarw.5' oligoadenylates and having increased activity, in comparison with the 2'.fwdarw.5' oligoadenylates induced in cells treated with interferon (BAGLIONI C. et coll., 1981, Analogs of (2'-5')oligo(A). Endonuclease activation and inhibition of protein synthesis in intact cells. The Journal of Biological Chemistry, vol. 256, n.degree. 7, p. 3, 253-3 257).
Various researches have been carried out to synthesise (enzymatically and/or chemically) modified analogs of 2'.fwdarw.5' oligoadenylates induced in cells treated with interferon, which would be resistant to the degradation actions, without losing their biological activity.
Among these researches, it is possible to cite enzymatic synthesis by means of 2'.fwdarw.5A synthetase of "cordycepine 2.fwdarw.5A" from 2' deoxyadenosine triphosphate (DOETSCH et coll., 1981).
Cordycepine has been considered as inhibiting the synthesis of the proteins in an acellular system and the corresponding dephosphorylated compound ("core" or "nucleus") has been considered as blocking the blastic transformation of human lymphocytes.
The results have however been disputed (CHAPEKAR M. S. et coll., 1983, Biochem. Res. Comm., 115, 137-143) and it seems that the effects observed have been caused by the accumulation of toxic degradation products of cordycepine.
It is also possible to cite, among the researches carried out, the chemical synthesis of an analog of the nucleus of 2'.fwdarw.5' oligoadenylates, in a xylose series (IMBACH J. L. et coll., 1981, Tetrahedron Letters, vol. 22. n.degree. 47, p. 4 699-4 702), named "xylo 2'-5'A". This analog is shown to present greater stability with respect to phosphodiesterases than the nucleus of unmodified 2'-5' oligoadenylates, an interesting activity with respect to a DNA virus, such as Herpes, but not with respect to RNA virus (EPPSTEIN D., et coll., 1983, Nature, 302, 723-724).
There can also be mentioned the chemical synthesis and the modification at its 2' terminal end of a 2'-5' oligoadenylate into a compound called "tailed 2'-5' A" in which a hexylamine chain has been associated with a morpholine nucleus, itself condensed by a phosphate group onto the OH group at the 2' position of the terminal ribose. This derivative is very stable with respect to phosphodiesterases and activates L endoribonuclease in an acellular system (IMAI J. et coll., 1982. J. Biochem. Chem., 257, 12 739-12 741), but its antiviral activity has not been established.
Among these researches, it is possible also to mention the chemical synthesis of modified derivatives of 2'-5' oligoadenylates such as the derivatives of 2'-5'A triphosphates (represented by the formula pppA2'p5'A2'p5A) in which the phosphorus atoms at the beta and gamma positions of the triphosphate group at the 5' position are separated by a methylene group.
Another modification to obtain modified 2'-5'A oligoadenylates relates to the replacement of a hydroxyl group at the 3' position by an OCH.sub.3 group either in the terminal adenosine, or in all the adenosines (J. A. J. DEN HARTOG et coll., 1981, J. Org. Chem., 46, 2 242-2 251).
However it is shown that these two latter groups of compounds were weakly active, even inactive and did not show satisfactory metabolic stability (cf. the reference mentioned above and BAGLIONI et coll., 1981, J. Biol. Chem., 256, 2 353-2 357).
Other analogs, such as 5'S-methylthiophosphorothioates have been synthesised. Certain of these analogs are revealed to be stable. But a priori, the apparent differences of properties of these analogs does not seem to permit their use on human cells for therapeutic purposes to be envisaged (HAUGH M. C., CAYLEY P. J. et coll., 1983, Europ. J. Biochem., 132, 77-84).
Investigations have also borne on the incidence of the modification of the one or more phosphate groups carried by the carbon at the 5' position of 2'-5' oligoadenylates with respect to antimitogenic activity (cf. TORRENCE et coll., 1983, J. Medicinal Chemistry 26, n.degree. 12, 1674-1678). The compounds prepared within the scope of these researches are shown to present antimitogenic activity, but it has been found that certain of them do not activate endoribonuclease L in an in vitro cellular system, which prevents the establishment of a correlation between antimitogenic activity and antiviral action.
Other similar oligonucleotides of (2'-5') (A).sub.m have been synthesised enzymatically by replacing the adenosine, particularly by 8-azaadenosine, toyocamycine, sangivamycine, formycine, 8-bromoadenosine, tubercidine and guanosine. It was shown that the majority of these compounds were degraded in cellular extracts. Only inhibition tests of the synthesis of proteins and of cellular proliferation have been carried out in intact cells, but the antiviral activity has not been established (B. G. HUGHES and R. K. ROBINS, 1983, Biochemistry, 22, n.degree.9, 2 127-2 135). None of the analogs of (2'-5')(A) synthesized hitherto have shown stability properties -both with respect to phosphodiesterases and phosphases-, and sufficient biological activity to be able to envisage using them in the therapeutic treatment of viral infections.